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EP2413961B1 - Method for prophylaxis of influenza using vaccine for intranasal administration - Google Patents

Method for prophylaxis of influenza using vaccine for intranasal administration Download PDF

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Publication number
EP2413961B1
EP2413961B1 EP10714104.6A EP10714104A EP2413961B1 EP 2413961 B1 EP2413961 B1 EP 2413961B1 EP 10714104 A EP10714104 A EP 10714104A EP 2413961 B1 EP2413961 B1 EP 2413961B1
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Prior art keywords
vaccine
antigen
dose
administration
composition
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EP10714104.6A
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German (de)
French (fr)
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EP2413961A1 (en
Inventor
Hideki Hasegawa
Sadao Manabe
Takeshi Tanimoto
Takashi Miyazaki
Taizou Kamishita
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Japan As Represented By Director-General Of Na
RESEARCH FOUNDATION FOR MICROBIAL DISEASES OF
Toko Yakuhin Kogyo KK
Original Assignee
Toko Yakuhin Kogyo KK
Research Foundation for Microbial Diseases of Osaka University BIKEN
National Institute of Infectious Diseases
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a vaccine for the prophylaxis of influenza, and a method for the prophylaxis of influenza.
  • Influenza is a viral infectious disease that repeatedly becomes epidemic with different antigen every year.
  • the vaccines approved in Japan for the prophylaxis of influenza are those for subcutaneous administration. Since the vaccination induces an IgG antibody having a neutralizing activity in the serum, it is highly effective for preventing progression of the condition into a more severe one such as pneumonia and the like.
  • IgA is the main prophylactic component. Since IgA is not induced by subcutaneous administration, the infection-preventive effect is not sufficient. Therefore, the development of an infection-preventive vaccine has been desired for a long time.
  • the present inventors have developed an influenza vaccine for mucosal administration comprising a double-stranded RNA and an influenza virus antigen, which is superior in the infection-preventive effect ( WO2005/014038 ).
  • the present invention provides the following.
  • the influenza virus includes any subtype known at present, and subtypes which will be isolated and identified in the future. Since no epidemic has been heretofore observed in human and human infection needs to be effectively prevented hereafter, the influenza virus is preferably a subtype consisting of a combination of a type selected from H1 - H16 excluding H1 and H3 (i.e., H2 and H4 - 16) and a type selected from N1 - N9. These subtypes are also called new type of influenza virus.
  • the aforementioned subtype is more preferably a subtype consisting of a combination of a type selected from H5, H7 and H9 and a type selected from N1 - N9.
  • the influenza virus may be one type of strain belonging to the same subtype, or two or more types of strains belonging to the same subtype, or two or more types of strains belonging to different subtypes.
  • influenza virus antigen contained in the vaccine composition of the present invention is largely divided into an inactivated antigen and a subunit antigen.
  • inactivated antigen refers to an antigen deprived of infectivity, used as a vaccine antigen; such antigens include, but are not limited to, complete virus particle virions, incomplete virus particles, virion-constituting particles, virus non-structural proteins, antigens that protect against infections, neutralizing reaction epitopes and the like.
  • inactivated antigen refers to an antigen deprived of infectivity, but retaining immunogenicity; when such an antigen is used as a vaccine, it is called an "inactivated vaccine.ā€
  • inactivation methods of antigens include, but are not limited to, physical (e.g., X-ray irradiation, heat, ultrasound), chemical (formalin, mercury, alcohol, chlorine) or other procedures.
  • Subunit antigen per se also falls within the definition of inactivated antigen because they have usually lost infectivity. Alternatively, a killed virus may be used.
  • subunit antigen refers to a component derived from an influenza virus.
  • the subunit antigen includes hemagglutinin (HA), neuraminidase (NA), matrices (Ml, M2), non-structures (NS), polymerases (PB1, PB2: basic polymerases 1 and 2, acidic polymerase (PA)), nuclear proteins (NP) and the like, with preference given to HA or NA, which is exposed to the surface of the virus particle.
  • HA hemagglutinin
  • NA neuraminidase
  • NS non-structures
  • PA acidic polymerase
  • NP nuclear proteins
  • the amount of the influenza virus antigen to be contained in the vaccine composition of the present invention is not particularly limited as long as it is sufficient to produce secretary IgA, and can be appropriately determined in consideration of the ratio to the below-mentioned poly (I:C) or a derivative thereof.
  • HA is used as an antigen, for example, its concentration is preferably 10 - 500 ā‡ g HA/mL (based on HA), more preferably 30 - 400 ā‡ g HA/mL (based on HA). The aforementioned concentration is obtained by measuring the concentration of HA protein.
  • Poly(I:C) as contained in the vaccine composition of the present invention is a double-stranded RNA (dsRNA) comprising polyinosinic acid (pI) and polycitidic acid (pC).
  • dsRNA double-stranded RNA
  • pI polyinosinic acid
  • pC polycitidic acid
  • a derivative of poly (I:C) is poly (I:C x U) or poly (I x U:C) (where x is on average a number from 3 to 40).
  • a derivative of poly (l:C) is poly (I:C 12 U) or poly (C:I 12 U), which is commercially available under the trade name AmpligenTM.
  • Poly (l:C) or a derivative thereof is supplied in a size sufficient to produce secretory IgA.
  • sizes include 100 bp or more, with preference given to sizes of 300 bp or more, which sizes, however, are not to be construed as limiting.
  • the upper limit of size include, but are not limited to, 10 8 bp.
  • Poly (I:C) or a derivative thereof is present at a concentration sufficient to produce secretory IgA.
  • concentration of poly (I:C) or a derivative thereof is, for example, 0.1 to 10 mg/mL, more preferably 0.5 to 2 mg/mL, and still more preferably about 1 mg/mL (e.g., 0.8 to 1.2 mg/mL).
  • the weight ratio of the influenza virus antigen and poly (I:C) or a derivative thereof to be contained in the vaccine composition of the present invention is recommended to be 1:1 - 1:50.
  • a carboxyvinyl polymer (CVP) contained in the vaccine composition of the present invention is a hydrophilic polymer which is produced by polymerization of acrylic acid as the main monomer component and includes conventional ones such as CarbopolTM commercially available from Lubrizol Advanced Materials, Inc. US and the like.
  • the concentration of CVP used in the present invention is generally in the range of 0.1 - 2.0 % by weight.
  • the vaccine composition of the present invention may contain a water-soluble basic substance for the purpose of thickening CVP.
  • the water-soluble basic substance includes, for example, inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonia, etc.), and organic bases such as alkylamines (e.g., methylamine, ethylamine, propylamine, etc.), dialkylamines (e.g., dimethylamine, diethylamine, dipropylamine, etc.), trialkylamines (e.g., trimethylamine, triethylamine, tripropylamine, etc.), alkanolamines (e.g., methanolamine, ethanolamine, propanolamine, etc.), dialkanolamines (e.g., dimethanolamine, diethanolamine, dipropanolamine, etc.), trialkanolamines (e.g., trimethanolamine, triethanolamine, tripropanolamine, etc.), amino acids
  • the pH value of the vaccine composition of the present invention is adjusted to the desired pH with a water-soluble basic substance or other pH adjustors taking into consideration the stability or absorption of an influenza virus antigen.
  • Preferable pH range is 6.0 - 8.0.
  • Adjustment of the viscosity can be performed depending on the vaccine dosage form. For example, CVP corresponding to 0.1 - 2.0 % by weight is thickened with a water-soluble basic substance and the viscosity is adjusted by applying an outside shearing force, or the viscosity is adjusted with a viscosity modulating agent and an outside shearing force, whereby a base managed to suit the spray angle and spray density from a sprayer is prepared. Thereafter, an influenza virus antigen and poly (I:C) are admixed.
  • the vaccine composition of the present invention may contain a suitable active medicament, diluent, bactericide, preservative, surfactant, stabilizer and the like which can be used together with the vaccine.
  • the present invention relates to a method of preventing influenza, comprising a step of administering a vaccine composition comprising an effective amount of an influenza virus antigen and poly (I:C) or a derivative thereof, and a carboxyvinyl polymer at least once to the nasal mucosa of a subject in need thereof.
  • the subject of administration of the vaccine composition of the present invention includes, but is not limited to, mammals including human, birds and the like.
  • Nasal mucosal administration of the vaccine composition can be performed in an appropriate form.
  • Various methods such as spraying, coating, or direct dripping of a vaccine liquid can be used.
  • Administration frequency of the vaccine composition of the present invention is at least once, preferably at least twice, in view of the effectiveness. Additional administration is sometimes called booster immunization. Booster immunization makes it possible to achieve a higher infection-protective effect.
  • the interval of booster immunization is recommended to be at least 1 week, preferably 1 to 4 weeks.
  • a vaccine corresponding to pandemic influenza is highly important and highly urgent as compared to vaccine for seasonal influenza, and needs to be preferentially developed. Therefore, a sample vaccine containing a vaccine for new type of influenza (PR8-IBCDC-RG2 strain: attenuated virus strain of A/Indo/5/2005 (H5N1)) and Ampligen in a 10-fold amount of HA antigen was prepared in the same manner as for seasonal influenza HA vaccine.
  • PR8-IBCDC-RG2 strain attenuated virus strain of A/Indo/5/2005 (H5N1)
  • Ampligen in a 10-fold amount of HA antigen was prepared in the same manner as for seasonal influenza HA vaccine.
  • the sample vaccine contained, as an additive for enhancing an immune response in the nasal mucosa by delaying the clearance of vaccine from the nasal mucosa, a CVP base [mixture of 0.55% CVP, 1.2% L-arginine, 1% glycerin] also applied to allergy medicines.
  • a nasal administration test 7-week-old female BALB/c mice were used (5 per group), and antigen was administered at 0.033 - 1 ā‡ g HA/head.
  • a group for which Ampligen was added to vaccine in a 10-fold amount of HA antigen and CVP base was not added
  • a group for which Ampligen was added to vaccine in a 20-fold amount of HA antigen and CVP base was not added
  • a group for which CVP alone was added to vaccine a group to which vaccine without addition was nasally administered
  • a group to which vaccine without addition was intramuscularly administered were prepared.
  • the vaccine was administered twice at 3-week intervals, and nasal washings and serum were recovered 2 weeks after booster.
  • the administration method was changed from conventional nasal drip using a micropipette to injection by inserting about 4 mm of a blunt needle (27G, 0.4 mmx38 mm) set to a microsyringe.
  • the optimal dose and administration frequency for mouse were examined based on the measurements of serum specific IgG antibody titer and HI antibody titer after addition of Ampligen, CVP and the like to an antigen, and the comparison with existing vaccines having known composition and known administration method, and using a "new type influenza virus antigen at high concentration".
  • New type influenza vaccine stock solution and virus for evaluation of cross-reactive antibody titer 1.
  • new type influenza vaccine stock solution lot No.; FPBMW0612-c (protein concentration: 3110 ā‡ g/mL, HA content 992 ā‡ g HA/mL, origin virus strain: A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2) (The Research Foundation for Microbial Diseases of Osaka University)
  • AmpligenĀ® containing Ampligen (2.5 mg/mL) manufactured by Hemispherx
  • Fig. 2 shows a schematic view of test flows.
  • a vaccine containing A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2 virus antigen obtained by adding Ampligen to a 20-fold concentration of HA antigen, and adding CVP base (1.1% carboxyvinyl polymer, 2.4% L-arginine, 2.0% glycerol) to 50.0 v/v% of the total amount
  • CVP base (1.1% carboxyvinyl polymer, 2.4% L-arginine, 2.0% glycerol
  • the transnasal administration group was administered with 6 ā‡ L into one nostril, and the control non-addition vaccine subcutaneous administration group was administered with 50 ā‡ L to unilateral femoral area.
  • nasal swab specific IgA-ELISA antibody titer and cross-reactive IgA-ELISA antibody titer:
  • Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to protein concentration of 1 ā‡ g/mL) was solid phased (4Ā°C, overnight) on a 96-well ELISA plate at 100 ā‡ L/well, and washed 3 times with PBS containing 0.1% Tween 20. A sample diluted 2-fold series or negative control was added at 100 ā‡ L/well. Then, the plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. An antibody for detection (alkaliphosphatase-labeled anti-mouse IgG or biotin-labeled anti-mouse IgA) was added at 100 ā‡ L/well.
  • the plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20 (for IgA detection, alkaliphosphatase-labeled streptavidin was added at 100 ā‡ L/well, incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20). Then, 4-nitrophenylphosphate (1 mg/mL, diluted with 0.1 M diethanolamine (pH 9.8)) was added at 100 ā‡ L/well, and the plate was shaded. After incubation at room temperature for 30 min, the absorbance at 405 nm was measured.
  • the maximum dilution rate that affords an absorbance of sample exceeding the average absorbance of negative control+2SD was taken as the antibody titer of the sample.
  • specific antibody titer the same strain as the administration vaccine antigen was used and, for cross-reactive antibody titer, vaccine antigen of a different strain was used.
  • HIā€¢ neutralizing antibody titer were measured in the same manner.
  • mice ā‡ , 6.5w when test was started
  • the antigen A/Indonesia/5/2005 (H5N1)
  • H5N1 antigen/Indonesia/5/2005
  • CVP was transnasally administered 1 - 4 times, using, as a comparison control, an antigen+Ampligen+CVP transnasal administration group for the first half of the test group and transnasal administration of antigen alone/intramuscular administration of antigen alone/saline transnasal administration for the latter half.
  • Tables 12-19 and Fig. 3 The results are shown in Tables 12-19 and Fig. 3 .
  • transnasal administration-type vaccines show lower specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer.
  • the vaccine evaluated at this time was found to acquire specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer equivalent to or above those of the vaccines with conventional composition and administration method, by the addition of AmpligenĀ® and the like.
  • Vaccine composition dose administration group vaccine stock solution( ā‡ L) Ampligen ( ā‡ L) C ā‡ /P ( ā‡ L) M/75PBS(pH72) ( ā‡ L) total (mL) note high dose (0.3 ā‡ g HA/dose) 1,4,7,10 302.42 2400.0 3000 297.58 6 for 1000 times (transnasal) moderate dose (0.1 ā‡ g HA/dose) 2,5,8,11 100.81 800.0 3000 2099.19 6 for 1000 times (transnasal) low dose (0.033 ā‡ g HA/dose) 3,6,9,12 33.27 264.0 3000 2702.73 6 for 1000 times (transnasal) high dose (0.3 ā‡ g HA/dose) 13,16,19,22 302.42 5697.58 6 for 1000 times (transnasal) moderate dose (0.1 ā‡ g HA/dose) 14,17,20
  • Influenza vaccine H5N1 strain stock solution (lot No., FPBMQI0813, protein concentration: 816 ā‡ g/mL, HA content 324 ā‡ g HA/mL, origin of virus strain: A/Bar-headed Goose/Qinghai/1A/2005 (H5N1))
  • H5N1 strain attenuated strain (clade 2.2) of A/Qinghai/1A/2005 (H5N1)
  • Fig. 4 shows outline of test flows.
  • Test group constitution group vaccine HA antigen ( ā‡ g/dose) Ampligen ( ā‡ g/ dose) Poly L-Arg ( ā‡ g/ dose) CVP addition head note 1 30 - 4 vaccine alone 2 30 600 O 4 trial vaccine 3 30 1500 O 4 Poly L-Arg 4 - - 1 control group
  • Anesthesized Cynomolgus monkeys (Macaca fascicularis) was transnasally administered with a vaccine containing A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) strain virus antigen or saline at 3 weeks intervals, using a transnasal administration device with trial actuator (test actuator : manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya, Spray Pump; Apta Pharma, VP-7 type, spraying use) six times by 150 ā‡ l (50 ā‡ l ā‡ 3) to each nasal cavity (total 300 ā‡ l), and samples (nasal swab and serum) were taken at the time of each administration, and every 2 weeks from the booster up to 12 weeks from the booster. Furthermore, where necessary, blood samplesā€¢nasal swab were taken every 2 weeks thereafter.
  • nasal swab specific IgA-ELISA antibody titer and IgA concentration
  • the nasal swab was measured for the total IgA concentration and the specific IgA-ELISA antibody titer calculated in the same manner as in Example 2 was amended to be the numerical value per total IgA concentration of 1 ā‡ g/mL.
  • serum specific IgG-ELISA antibody titer, specific, cross-reactive HI (hemagglutination inhibition) antibody titer, specific/cross-reactive neutralizing antibody titer
  • the outline of the measurement method of the ELISA antibody titer was as described below.
  • Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to protein concentration of 1 ā‡ g/mL) was solid phased (4Ā°C, overnight) on a 96-well ELISA plate at 100 ā‡ L/well, and washed 3 times with PBS containing 0.1% Tween 20. A sample diluted 2-fold series or negative control was added at 100 ā‡ L/well. Then, the plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. An antibody for detection (alkaliphosphatase-labeled anti-monkey IgG or biotin-labeled anti-monkey IgA) was added at 100 ā‡ L/well.
  • the plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20.
  • the substrate solution (4-NPP or TMB) was added at 100 ā‡ L/well and the mixture was shaded and incubated at room temperature for 30 min.
  • a stop solution (2M H 2 SO 4 solution for 4-NPP or 650 nm stop solution for TMB) was added, and the absorbance (405 nm or 655 nm) was measured.
  • the maximum dilution rate that affords an absorbance of sample exceeding the average absorbance of negative control+2SD was taken as the antibody titer of the sample.
  • specific antibody titer the same strain as the administration vaccine antigen was used and, for cross-reactive antibody titer, vaccine antigen of a different strain was used.
  • HI antibody titer and neutralizing antibody titer were measured in the same manner.
  • specific HI (hemagglutination inhibition) antibody titer and cross-reactive HI antibody titer They were measured in the same manner as in Example 2.
  • specific neutralizing antibody titer and cross-reactive neutralizing antibody titer They were measured in the same manner as in Example 2.
  • transnasal spray administration of a vaccine containing Ampligen and CVP to monkey induces specific IgA production in the mucous membrane, and derivation of specific neutralizing antibody in the serum. Since these effects were insufficient with an additive-free vaccine, the usefulness of addition of Ampligen and CVP was confirmed.

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Description

    Technical Field
  • The present invention relates to a vaccine for the prophylaxis of influenza, and a method for the prophylaxis of influenza.
    • Background Art
  • Influenza is a viral infectious disease that repeatedly becomes epidemic with different antigen every year. The vaccines approved in Japan for the prophylaxis of influenza are those for subcutaneous administration. Since the vaccination induces an IgG antibody having a neutralizing activity in the serum, it is highly effective for preventing progression of the condition into a more severe one such as pneumonia and the like. In the upper airway mucosa, which is the infection site, however, IgA is the main prophylactic component. Since IgA is not induced by subcutaneous administration, the infection-preventive effect is not sufficient. Therefore, the development of an infection-preventive vaccine has been desired for a long time.
  • The present inventors have developed an influenza vaccine for mucosal administration comprising a double-stranded RNA and an influenza virus antigen, which is superior in the infection-preventive effect ( WO2005/014038 ).
    • Disclosure of the Invention
  • It is therefore an object of the present invention to provide a novel vaccine which is useful for the prophylaxis of influenza, and a method for the prophylaxis of influenza.
  • To achieve the.above-mentioned object, the present inventors have tried to examine the effects of vaccines by adding various components, and completed the present invention.
  • Accordingly, the present invention provides the following.
    1. [1] A vaccine composition for nasal mucosal administration, comprising (i) an influenza virus antigen, (ii) polyriboinosinic polyribocytidylic acid (poly (I:C)), poly (I:CxU) or poly (IxU:C), and (iii) a carboxyvinyl polymer, wherein x is on average a number from 3 to 40.
    2. [2] The composition of [1], wherein the aforementioned antigen is a subunit antigen or an inactivated antigen.
    3. [3] The composition of [2], wherein the aforementioned subunit antigen comprises at least one subunit antigen selected from the group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza virus.
    4. [4] The composition of [2], wherein the aforementioned subunit antigen comprises at least one subunit antigen selected from the group consisting of HA and NA.
    5. [5] The composition of [1], wherein the aforementioned poly (I:CxU) is poly (I:C12U).
    6. [6] A vaccine composition comprising an effective amount of (i) an influenza virus antigen, (ii) poly (I:C), poly (I:CxU) or poly (IxU:C), and (iii) a carboxyvinyl polymer, wherein x is on average a number from 3 to 40, said composition being for use in a method of preventing influenza, comprising a step of administering said composition at least once to the nasal mucosa of a subject in need thereof.
    7. [7] The composition for use according to [6], wherein the aforementioned antigen is a subunit antigen or an inactivated antigen.
    8. [8] The composition for use according to [7], wherein the aforementioned subunit antigen comprises at least one subunit antigen selected from the group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza virus.
    9. [9] The composition for use according to [7], wherein the aforementioned subunit antigen comprises at least one subunit antigen selected from the group consisting of HA and NA.
    10. [10] The composition for use according to [6], wherein the aforementioned poly (I:CxU) is poly (I:C12U).
    11. [11] The composition for use according to [6], wherein the aforementioned method comprises administering the composition at least twice.
    12. [12] The composition for use according to [11], wherein the aforementioned
      method comprises administering the composition at an interval of at least 1 week.
  • The characteristics and the advantages of the present invention will be apparent from the detailed description of the invention below.
    • Brief Description of the Drawings
    • Figure 1 shows an immune response in a nasal administration test of an influenza vaccine comprising Ampligen and a CVP base in combination.
    • Figure 2 is a flow chart of mouse immunity test in Example 2.
    • Figure 3 is a summary of the vaccine administration test in Example 2.
    • Figure 4 is a flow chart of monkey immunity test in Example 3.
    • Figure 5 shows the results of Serum IgG ELISA in Example 3.
    • Figure 6 shows the results of nasal swab IgA ELISA in Example 3.
    • Figure 7 shows a summary of the vaccine administration test in Example 3.
    Detailed Description of the Invention
  • In the present invention, the influenza virus includes any subtype known at present, and subtypes which will be isolated and identified in the future. Since no epidemic has been heretofore observed in human and human infection needs to be effectively prevented hereafter, the influenza virus is preferably a subtype consisting of a combination of a type selected from H1 - H16 excluding H1 and H3 (i.e., H2 and H4 - 16) and a type selected from N1 - N9. These subtypes are also called new type of influenza virus. The aforementioned subtype is more preferably a subtype consisting of a combination of a type selected from H5, H7 and H9 and a type selected from N1 - N9. The influenza virus may be one type of strain belonging to the same subtype, or two or more types of strains belonging to the same subtype, or two or more types of strains belonging to different subtypes.
  • The influenza virus antigen contained in the vaccine composition of the present invention is largely divided into an inactivated antigen and a subunit antigen.
  • The term "inactivated antigen" as used herein refers to an antigen deprived of infectivity, used as a vaccine antigen; such antigens include, but are not limited to, complete virus particle virions, incomplete virus particles, virion-constituting particles, virus non-structural proteins, antigens that protect against infections, neutralizing reaction epitopes and the like. The term "inactivated antigen" as used herein refers to an antigen deprived of infectivity, but retaining immunogenicity; when such an antigen is used as a vaccine, it is called an "inactivated vaccine." Examples of the inactivation methods of antigens include, but are not limited to, physical (e.g., X-ray irradiation, heat, ultrasound), chemical (formalin, mercury, alcohol, chlorine) or other procedures. Subunit antigen per se also falls within the definition of inactivated antigen because they have usually lost infectivity. Alternatively, a killed virus may be used.
  • The term "subunit antigen" as used herein refers to a component derived from an influenza virus. The subunit antigen includes hemagglutinin (HA), neuraminidase (NA), matrices (Ml, M2), non-structures (NS), polymerases (PB1, PB2: basic polymerases 1 and 2, acidic polymerase (PA)), nuclear proteins (NP) and the like, with preference given to HA or NA, which is exposed to the surface of the virus particle. Currently known types of HA are HA1 to HA16, and known types of NA are NA1 to NA9. The subunit antigen may be purified from a pathogen such as a naturally occurring virus, or may be prepared by a synthetic or recombinant technology. Such methods are well known and in common use in the art, and can be performed using commercially available equipment, reagents, vectors and the like.
  • The amount of the influenza virus antigen to be contained in the vaccine composition of the present invention is not particularly limited as long as it is sufficient to produce secretary IgA, and can be appropriately determined in consideration of the ratio to the below-mentioned poly (I:C) or a derivative thereof. When HA is used as an antigen, for example, its concentration is preferably 10 - 500 Āµg HA/mL (based on HA), more preferably 30 - 400 Āµg HA/mL (based on HA). The aforementioned concentration is obtained by measuring the concentration of HA protein.
  • Poly(I:C) as contained in the vaccine composition of the present invention is a double-stranded RNA (dsRNA) comprising polyinosinic acid (pI) and polycitidic acid (pC).
  • A derivative of poly (I:C) is poly (I:CxU) or poly (IxU:C) (where x is on average a number from 3 to 40). Preferably, a derivative of poly (l:C) is poly (I:C12U) or poly (C:I12U), which is commercially available under the trade name Ampligenā„¢.
  • Poly (l:C) or a derivative thereof is supplied in a size sufficient to produce secretory IgA. Examples of such sizes include 100 bp or more, with preference given to sizes of 300 bp or more, which sizes, however, are not to be construed as limiting. Examples of the upper limit of size include, but are not limited to, 108 bp.
  • Poly (I:C) or a derivative thereof is present at a concentration sufficient to produce secretory IgA. Such a concentration of poly (I:C) or a derivative thereof is, for example, 0.1 to 10 mg/mL, more preferably 0.5 to 2 mg/mL, and still more preferably about 1 mg/mL (e.g., 0.8 to 1.2 mg/mL).
  • The weight ratio of the influenza virus antigen and poly (I:C) or a derivative thereof to be contained in the vaccine composition of the present invention is recommended to be 1:1 - 1:50.
  • A carboxyvinyl polymer (CVP) contained in the vaccine composition of the present invention is a hydrophilic polymer which is produced by polymerization of acrylic acid as the main monomer component and includes conventional ones such as Carbopolā„¢ commercially available from Lubrizol Advanced Materials, Inc. US and the like. The concentration of CVP used in the present invention is generally in the range of 0.1 - 2.0 % by weight.
  • The vaccine composition of the present invention may contain a water-soluble basic substance for the purpose of thickening CVP. The water-soluble basic substance includes, for example, inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonia, etc.), and organic bases such as alkylamines (e.g., methylamine, ethylamine, propylamine, etc.), dialkylamines (e.g., dimethylamine, diethylamine, dipropylamine, etc.), trialkylamines (e.g., trimethylamine, triethylamine, tripropylamine, etc.), alkanolamines (e.g., methanolamine, ethanolamine, propanolamine, etc.), dialkanolamines (e.g., dimethanolamine, diethanolamine, dipropanolamine, etc.), trialkanolamines (e.g., trimethanolamine, triethanolamine, tripropanolamine, etc.), amino acids (e.g., arginine, lysine, ornithine, etc.) and the like. These water-soluble bases are used in an amount which is necessary for neutralization to adjust the pH value of CVP aqueous solution to a desired pH.
  • The pH value of the vaccine composition of the present invention is adjusted to the desired pH with a water-soluble basic substance or other pH adjustors taking into consideration the stability or absorption of an influenza virus antigen. Preferable pH range is 6.0 - 8.0.
  • Adjustment of the viscosity can be performed depending on the vaccine dosage form. For example, CVP corresponding to 0.1 - 2.0 % by weight is thickened with a water-soluble basic substance and the viscosity is adjusted by applying an outside shearing force, or the viscosity is adjusted with a viscosity modulating agent and an outside shearing force, whereby a base managed to suit the spray angle and spray density from a sprayer is prepared. Thereafter, an influenza virus antigen and poly (I:C) are admixed.
  • The vaccine composition of the present invention may contain a suitable active medicament, diluent, bactericide, preservative, surfactant, stabilizer and the like which can be used together with the vaccine.
  • The present invention relates to a method of preventing influenza, comprising a step of administering a vaccine composition comprising an effective amount of an influenza virus antigen and poly (I:C) or a derivative thereof, and a carboxyvinyl polymer at least once to the nasal mucosa of a subject in need thereof.
  • The subject of administration of the vaccine composition of the present invention includes, but is not limited to, mammals including human, birds and the like.
  • Nasal mucosal administration of the vaccine composition can be performed in an appropriate form. Various methods such as spraying, coating, or direct dripping of a vaccine liquid can be used.
  • Administration frequency of the vaccine composition of the present invention is at least once, preferably at least twice, in view of the effectiveness. Additional administration is sometimes called booster immunization. Booster immunization makes it possible to achieve a higher infection-protective effect. The interval of booster immunization is recommended to be at least 1 week, preferably 1 to 4 weeks.
  • The present invention is hereinafter described in more detail by means of the following examples, which, however, are not to be construed as limiting the present invention.
    • (Example 1)
  • Nasal administration test of whole virion vaccine containing Ampligen and CVP in combination for new type of influenza, using mouse
  • In the development of an influenza vaccine for nasal administration, a vaccine corresponding to pandemic influenza is highly important and highly urgent as compared to vaccine for seasonal influenza, and needs to be preferentially developed. Therefore, a sample vaccine containing a vaccine for new type of influenza (PR8-IBCDC-RG2 strain: attenuated virus strain of A/Indo/5/2005 (H5N1)) and Ampligen in a 10-fold amount of HA antigen was prepared in the same manner as for seasonal influenza HA vaccine. The sample vaccine contained, as an additive for enhancing an immune response in the nasal mucosa by delaying the clearance of vaccine from the nasal mucosa, a CVP base [mixture of 0.55% CVP, 1.2% L-arginine, 1% glycerin] also applied to allergy medicines.
  • For a nasal administration test, 7-week-old female BALB/c mice were used (5 per group), and antigen was administered at 0.033 - 1 Āµg HA/head. As a comparison control, a group for which Ampligen was added to vaccine in a 10-fold amount of HA antigen and CVP base was not added, a group for which Ampligen was added to vaccine in a 20-fold amount of HA antigen and CVP base was not added, a group for which CVP alone was added to vaccine, a group to which vaccine without addition was nasally administered, and a group to which vaccine without addition was intramuscularly administered were prepared. The vaccine was administered twice at 3-week intervals, and nasal washings and serum were recovered 2 weeks after booster. Specific IgA-ELISA antibody titer of the nasal washings was measured, and the antibody titers of hemagglutination inhibition (HI), neutralizing antibody and specific IgG-ELISA of the serum were measured. Since the sample vaccine had high viscosity, the administration method was changed from conventional nasal drip using a micropipette to injection by inserting about 4 mm of a blunt needle (27G, 0.4 mmx38 mm) set to a microsyringe.
  • The results are shown in Tables 1 - 9. [Table 1]
    A/Hiroshima HA vaccine+Poly (I:C) or Ampligen (lot comparison)
    group antigen administration route dose numbers/ group antigen (Āµg HA or ptn) Poly (I:C) Ampligen (new)
    1 A/Hiroshima /52/2005 (H3N2) HA vaccine in 2 Āµl to each nasal cavity 5 0.1
    2 0.1 0.1
    3 0.1 1
    4 2.7 Āµl to each nasal cavity 0.1 10
    5 0.1 0.1
    6 0.1 1
    7 0.1 10
    8 SC 100 0.1
    9 whole virion in 0.2
    10 none (control) 10
    • 1st:1/28 BALB/c mice (7wā™€)
    • 2nd:2/18
    • Sampling:2/28
    • Antigen was A/Hiroshima/52/2005 (H3N2) HA vaccine FTHAG0712 (1205 Āµg HA/mL) or whole virion vaccine (344 Āµg ptn/mL)
    • poly(I:C) [SIGMA P-0913] was adjusted to 1.88 mg/mL Ampligen (new) was Lot0701HE
    [Table 2]
    exp. A/Hiroshima HA vaccine+Poly (I:C) or Ampligen NW IgA ELISA
    group 1 2 3 4 5 6 7 8 9 10
    antigen A/Hiroshima /52/2005 (H3N2) HA vaccine whole virion none (control)
    administration route in sc in
    dose 2 Āµl to each nostril (nasal cavity) 2.7 Āµl to each nostril (nasal cavity) 2 Āµl to each nostril (nasal cavity) 100 Āµl 2 Āµl to each nostril (nasal cavity)
    numbers/group 5 10
    antigen (Āµg HA or ptn) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2
    adjuvant (Āµg/head) Poly (I:C) 0.1 1 10
    Ampligen (new) 0.1 1 10
    animal No. 1 1 1 1 16 1 1 128 1 64 1
    2 1 32 16 16 128 4 32 1 8 1
    3 1 2 16 128 32 32 4 1 8 1
    4 1 16 32 256 32 8 16 1 32 1
    5 1 64 32 64 2 2 16 1 32 1
    geometric mean 1.0 9.2 12.1 55.7 12.1 4.6 21.1 1.0 21.1 1.0
    SD 0.0 26.2 13.0 100.6 52.0 12.9 50.6 0.0 23.0 0.0
    geometric mean titer (GMT,2^n) 0.0 3.2 3.6 5.8 3.6 2.2 4.4 0.0 4.4 0.0
    positive conversion ratio (ā‰„32,%) 0.0 40.0 40.0 60.0 60.0 20.0 40.0 0.0 60.0 0.0
    [Table 3]
    exp. A/Hiroshima HA vaccine+Poly (I:C) or Ampligen Serum IgG
    group
    1 2 3 4 5 6 7 8 9 10
    antigen A/Hiroshima /52/2005 (H3N2) HA vaccine FTHAG0712 whole virion none (control)
    administration route in sc in
    dose 2 Āµl to each nostril (nasal cavity) 2.7 Āµl to each nostril (nasal cavity) 2 Āµl to each nostril (nasal cavity) 100 Āµl 2 Āµl to each nostril (nasal cavity)
    numbers/group 5 10
    antigen (Āµg HA or ptn) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2
    Poly (I:C) 0.1 1 10
    Ampligen (new) 0.1 1 10
    animal No. 1 50 50 ^100 6400 100 50 1600 51200 1600 50
    2 50 800 200 400 400 400 800 51200 400 50
    3 50 400 1600 6400 6400 1600 100 51200 200 50
    4 50 200 800 3200 200 6400 50 25600 400 100
    5 50 400 400 800 200 50 800 25600 1600 50
    geometric mean 50.0 263.9 400.0 2111.2 400.0 400.0 348.2 38802.3 606.3 53.6
    SD 0.0 282.0 609.9 2906.5 2763.7 2703.5 634.0 14021.7 698.6 15.8
    geometric mean titer (GMT,2^n) 5.6 8.0 8.6 11.0 8.6 8.6 8.4 15.2 9.2 5.7
    [Table 4]
    exp. A/Hiroshima HA vaccine+Poly (I:C) or Ampligen Serum HI
    group 1 2 3 4 5 6 7 8 9 10
    antigen A/Hiroshima /52/2005 (H3N2) HA vaccine whole virion none (control)
    administration route in sc in
    dose 2 Āµl to each nostril (nasal cavity) 2.7 Āµl to each nostril (nasal cavity) 2 Āµl to each nostril (nasal cavity) 100 Āµl 2 Āµl to each nostril (nasal cavity)
    numbers/group 5 10
    antigen (Āµg HA or ptn) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2
    Poly (I:C) 0.1 1 10
    Ampligen (new) 0.1 1 10
    animal No. 1 5 5 5 40 5 5 5 160 5 5
    2 5 5 5 5 5 5 5 320 5 5
    3 5 5 5 40 5 5 5 320 5 5
    4 5 5 5 20 5 10 5 160 5 5
    5 5 5 5 5 5 10 5 80 5 5
    geometric mean 5.0 5.0 5.0 15.2 5.0 6.6 5.0 183.8 5.0 5.0
    SD 0.0 0.0 0.0 17.5 0.0 2.7 0.0 107.3 0.0 ĀØ0.0
    geometric mean titer (GMT,2^n) 2.3 2.3 2.3 3.9 2.3 2.7 2.3 7.5 2.3 2.3
    seroconversion rate (ā‰„40,%) 0.0 0.0 0.0 40.0 0.0 0.0 0.0 100.0 0.0 0.0
    [Table 5]
    Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008.9.25) test group constitution
    group antigen (Āµg/dose) Ampligen (Āµg/dose) CVP (%) n administration route dose (Āµl) note
    1 0.3 3.00 5 in 2.0 antigen:Ampligen= 1:10
    2 0.1 1.00 5 in 2.0
    3 0.033 0.33 5 in 2.0
    4 0.3 3.00 0.55 5 in 2.0 antigen:Ampligen= 1:10 + CVP
    5 0.1 1.00 0.55 5 in 2.0
    6 0.033 0.33 0.55 5 in 2.0
    7 0.3 6.00 5 in 2.0 antigen:Ampligen= 1:10
    8 0.1 2.00 5 in 2.0
    9 0.033 0.67 5 in 2.0
    10 0.3 0.55 5 in 2.0 antigen + CVP
    11 0.1 0.55 5 in 2.0
    12 0.033 0.55 5 in 2.0
    13 0.3 5 in 2.0 antigen alone
    14 0.1 5 in 2.0
    15 0.033 5 in 2.0
    16 0.3 5 im 50.0 antigen alone, intramuscular control
    17 10 in 2.0 saline control
    • 2008.9.30 1st immunization
    • 2008.10.21 2nd immunization
    • 2008.11.4 Sampling
    • Antigen was attenuated virus strain of PR8-IBCDC-RG2
    • strain:A/Indo/5/2005(H5N1), Lot FPBMW0612-0 3110 Āµg PTN/mL. about 1048 Āµg HA/mL))
    • Ampligen is manufactured by Hemispherx, Lot R-01:45.7 mg/mL
    • CVP is manufactured by Toko Yakuhin Kogyo, Lot No.INF-9B8, base for preparation (#03-01)
    • Remark 1: prepared for 300 mice
    • antigen: 300 mice X 0.3 Āµg/head/1.048 Āµg/Āµl=85.9 Āµl BALB/c mice, 7w ā™€, were used.
    [Table 6]
    Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008.9.25-) NW IgA (2008.11.6)
    group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
    antigen (Āµg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3
    Ampligen (Āµg/dose) 3.00 1.00 0.33 3.00 1.00 0.33 6.00 2.00 0.67
    CVP (%) 0.55 0.55 0.55 0.55 0.55 0.55
    administration method in in in in in in in in in in in in in in in im in
    animal No. 1 32 32 4 256 128 16 8 8 4 16 32 8 16 16 1 1 1
    2 16 16 8 512 128 16 32 4 4 32 2 2 4 8 8 1 1
    3 32 32 4 128 128 2 16 8 2 16 2 1 16 2 4 1 2
    4 16 16 2 64 512 64 32 32 2 64 2 2 8 4 16 2 2
    5 8 32 1 64 64 64 64 8 8 4 1 4 8 64 2 2 2
    geometric mean titer (GMT) 18.4 24.3 3.0 147.0 147.0 18.4 24.3 9.2 3.5 18.4 3.0 2.6 9.2 9.2 4.0 1.3 1.5
    mean titer (2^n) 4.2 4.6 1.6 7.2 7.2 4.2 4.6 3.2 1.8 4.2 1.6 1.4 3.2 3.2 2.0 0.4 0.6
    positive conversion ratio (titerā‰„32, %) 40 60 0 100 100 40 60 20 0 40 20 0 0 20 0 0 0
    [Table 7]
    Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008.9.25-) Serum NT (2008.11.19)
    group 4 5 6 13 16
    antigen (Āµg/dose) 0.3 0.1 0.033 0.3 0.3
    Ampligen (Āµg/dose) 3.00 1.00 0.33
    CVP(%) 0.55 0.55 0.55
    Poly L-Arg (Āµg/dose)
    administration method in in in in im
    animal No. 1 80 160 80 20 160
    2 1280 10 40 10 80
    3 80 160 40 10 320
    4 160 160 160 10 80
    5 160 160 40 10 640
    geometric mean titer (GMT) 183.8 91.9 60.6 11.5 183.8
    mean titer (2^n) 7.5 6.5 5.9 3.5 7.5
    [Table 8]
    Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008.9.25-) Serum HI (2008.11)
    group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
    antigen (Āµg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3
    Ampligen (Āµg/dose) 3.00 1.00 0.33 3.00 1.00 0.33 6.00 2.00 0.67
    CVP (%) 0.55 0.55 0.55 0.55 0.55 0.55
    administration method in in in in in in in in in in in in in in in im in
    animal No. 1 5 5 5 5 10 5 5 5 5 5 5 5 5 5 5 10 5
    2 5 5 5 40 5 5 5 5 5 5 5 5 5 5 5 5 5
    3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 20 5
    4 5 5 5 10 10 20 10 5 5 5 5 5 5 5 5 5 5
    5 5 5 5 5 5 5 10 5 5 5 5 5 5 5 5 10 5
    geometric mean titer (GMT) 5.0 5.0 5.0 8.7 6.6 6.6 6.6 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 8.7 5.0
    mean titer (2^n) 2.3 2.3 2.3 3.1 2.7 2.7 2.7 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 3.1 2.3
    seroconversion rate (ā‰„40.%) 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 0
    [Table 9]
    Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008.9.25-) Serum IgG (2008.11.6)
    group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
    antigen (Āµg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3
    Ampligen (Āµg/dose) 3.00 1.00 0.33 3.00 1.00 0.33 6.00 2.00 0.67
    CVP (%) 0.55 0.55 0.55 0.55 0.55 0.55
    administration method in in in in in in in in in in in in in in in im in
    animal No. 1 1600 1600 400 12800 6400 1600 6400 200 50 800 400 400 1600 200 50 12800 50
    2 1600 1600 400 51200 6400 800 6400 800 800 6400 50 50 3200 3200 200 12800 50
    3 800 800 200 6400 12800 1600 6400 800 100 51200 50 100 1600 50 200 25600 50
    4 800 400 200 6400 25600 3200 6400 1600 100 800 200 100 3200 1600 400 12800 50
    5 800 400 200 12800 12800 3200 12800 800 1600 800 100 200 1600 3200 50 12800 50
    geometric mean titer (GMT) 1055.6 800.0 263.9 12800.0 11143.0 1837.9 7351.7 696.4 229.7 2785.8 114.9 132.0 2111.2 696.4 132.0 14703.3 50.0
    mean titer (2^n) 10.0 9.6 8.0 13.6 13.4 10.8 12.8 9.4 7.8 11.4 6.8 7.0 11.0 9.4 7.0 13.8 5.6
    positive conversion ratio (titerā‰„32, %) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 200
    (Results) Nasal administration test of whole virion vaccine for new type of influenza, containing Ampligen and CVP base in combination, and using mouse
  • A remarkable immunoenhancing effect was confirmed in the mucosa and serum in the group administered with a vaccine containing Ampligen and a CVP base in combination, which was not found in the group administered with Ampligen alone. The specific IgG, HI and neutralizing antibody titer (NT) of the serum increased to levels equivalent to those of the intramuscular injection group (no adjuvant).
  • A sample pandemic influenza vaccine containing, as an additive for enhancing an immune response in the nasal mucosa, a CVP base also applied to allergy medicines in combination, was prepared and used for a nasal administration test.
  • We investigated the convenience of nasal administration method at this time and obtained unexpected effects of a remarkably improved immune response of the nasal mucosa, a specific antibody titer of the serum, which is equivalent to that of intramuscular injection without adjuvant, and the like. Hence, a more practical vaccine was obtained.
    • (Example 2) Evaluation system via transnasal immunity in mouse (consideration of optimal dose and administration frequency for mouse)
  • The optimal dose and administration frequency for mouse were examined based on the measurements of serum specific IgG antibody titer and HI antibody titer after addition of Ampligen, CVP and the like to an antigen, and the comparison with existing vaccines having known composition and known administration method, and using a "new type influenza virus antigen at high concentration".
    • (Materials) 1. New type influenza vaccine stock solution and virus for evaluation of cross-reactive antibody titer:
  • new type influenza vaccine stock solution: lot No.; FPBMW0612-c (protein concentration: 3110 Āµg/mL, HA content 992 Āµg HA/mL, origin virus strain: A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2) (The Research Foundation for Microbial Diseases of Osaka University)
    • 2. Original strain of virus and inactivated vaccine antigen:
  • for measurement of specific antibody titer:
    • A/Indo/5/2005(H5N1)/PR8-IBCDC-RG2 strain: attenuated strain (clade 2.1) of A/Indonesia/5/2005 (H5N1)
    for measurement of cross-reactive antibody titer:
    • A/Anhui/01/2005(H5N1)/PR8-IBCDC-RG5 strain: attenuated strain (clade 2.3) of A/Anhui/01/2005 (H5N1)
    • A/bar-headed goose/Qinghai/lA/2005 strain: attenuated strain (clade 2.2) of A/Qinghai/1A/2005 (H5N1)
    • NIBRG-14 strain: attenuated strain (clade 1) of A/Vietnam/1194/2004 (H5N1)
    3. Adjuvant:
  • AmpligenĀ®: containing Ampligen (2.5 mg/mL) manufactured by Hemispherx
    • 4. Test method
  • Fig. 2 shows a schematic view of test flows.
  • A vaccine containing A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2 virus antigen (obtained by adding Ampligen to a 20-fold concentration of HA antigen, and adding CVP base (1.1% carboxyvinyl polymer, 2.4% L-arginine, 2.0% glycerol) to 50.0 v/v% of the total amount) was transnasally administered to BALB/c mice and samples (nasal swab and serum) were taken 3 weeks later. In this case, samples were not taken from a part of the mice, and booster was given thereto one to three times. The interval between the boosters for mice subjected to two or three times of boosters was 2 weeks. Samples were taken at 2 weeks from the last booster, and immune response was confirmed when all samples were taken. As comparison controls for the confirmation of the effects of Ampligen and CVP base added, a non-addition vaccine transnasal administration group (high dose, medium dose, low dose) with the same antigen dose but free of Ampligen and CVP base and a negative control group (transnasal administration (once) of saline) were set, and a non-addition vaccine intramuscular administration group (high dose) was set for comparison with vaccines having known dosage form and known administration method. From the above-mentioned test, the relationship between dose and administration frequency, and immune response was evaluated. In addition, immune response with a virus strain having different antigenicity was also investigated to obtain findings as to the cross-reactivity.
    • 5. Animals and vaccine (Table 10)
    • test animal: BALB/c mouse (ā™€, 6-week-old when the test was started), 10 mice/group
      trial vaccine for test consideration and adjuvant dose:
      • high dose group: [0.3 Āµg HA + 6 Āµg Ampligen]/dose
      • medium dose group: [0.1 Āµg HA + 2 Āµg Ampligen]/dose
      • low dose group: [0.033 Āµg HA + 0.66 Āµg Ampligen]/dose
  • As negative control, a saline administration group was set. In addition, for confirmation of effects of vaccine addition, non-addition vaccine transnasal administration group (high dose, medium dose, low dose) with the same antigen dose but free of Ampligen and CVP base was set, and an intramuscular administration group (high dose) was set for the comparison with conventional vaccines (known dosage formā€¢administration method). vaccine composition:
    • high dose group: [vaccine 0.3 ĀµL + Ampligen 2.4 ĀµL + CVP base 2.5 ĀµL]/dose
    • medium dose group: [vaccine 0.1ĀµL + Ampligen 0.8 ĀµL + M/75 PBS (PH 7.2) 1.6 ĀµL + CVP base 2.5 ĀµL]]/dose
    • low dose group: [vaccine 0.033 ĀµL + Ampligen 0.26 ĀµL + M/75 PBS (PH 7.2) 2.207 ĀµL + CVP base 2.5 ĀµL]/dose
      The vaccine for non-addition transnasal administration used as a negative control was obtained by removing Ampligen and CVP base from the above-mentioned composition, and adding M/75 PBS (pH 7.2) in a volume corresponding thereto. In addition, the vaccine for non-addition intramuscular administration was [vaccine 0.3 ĀµL + M/75 PBS (pH 7.2) 49.7 ĀµL]/dose.
    • Vaccine administration volume: (Table 11)
  • The transnasal administration group was administered with 6 ĀµL into one nostril, and the control non-addition vaccine subcutaneous administration group was administered with 50 ĀµL to unilateral femoral area.
    • 6. Measurement items (Tables 12-19)
  • nasal swab: specific IgA-ELISA antibody titer and cross-reactive IgA-ELISA antibody titer:
    • serum specific IgG-ELISA antibody titer, specific HI (hemagglutination inhibition) antibody titer and cross-reactive HI antibody titer, specific neutralizing antibody titer and cross-reactive neutralizing antibody titer were measured. Among the above-mentioned measurement items, the outline of the measurement method of the ELISA antibody titer was as described below.
  • Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to protein concentration of 1 Āµg/mL) was solid phased (4Ā°C, overnight) on a 96-well ELISA plate at 100 ĀµL/well, and washed 3 times with PBS containing 0.1% Tween 20. A sample diluted 2-fold series or negative control was added at 100 ĀµL/well. Then, the plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. An antibody for detection (alkaliphosphatase-labeled anti-mouse IgG or biotin-labeled anti-mouse IgA) was added at 100 ĀµL/well. The plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20 (for IgA detection, alkaliphosphatase-labeled streptavidin was added at 100 ĀµL/well, incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20). Then, 4-nitrophenylphosphate (1 mg/mL, diluted with 0.1 M diethanolamine (pH 9.8)) was added at 100 ĀµL/well, and the plate was shaded. After incubation at room temperature for 30 min, the absorbance at 405 nm was measured.
  • The maximum dilution rate that affords an absorbance of sample exceeding the average absorbance of negative control+2SD was taken as the antibody titer of the sample. For specific antibody titer, the same strain as the administration vaccine antigen was used and, for cross-reactive antibody titer, vaccine antigen of a different strain was used. In the following, HIā€¢ neutralizing antibody titer were measured in the same manner. specific HI (hemagglutination inhibition) antibody titer and cross-reactive HI antibody titer:
    • The test was performed according to the description in the "pathogen detection manual" (edited by National Institute of infectious Diseases). The red blood cells used were derived from chicken.
    specific neutralizing antibody titer and cross-reactive neutralizing antibody titer:
    • The test was performed according to the description in the "pathogen detection manual" (edited by National Institute of infectious Diseases). The culture after virus infection was performed for 4 days. In addition, a method comprising staining the cells with 0.1% Naphtol Blue Black/0.1% sodium acetate/9% acetic acid, drying the cell culture plate, adding 0.1M sodium hydroxide and measuring the absorbance at 630 nm was used for distinction between virus infectious cells and non-infectious cells.
    (Results)
  • BALB/c mice (ā™€, 6.5w when test was started) were used, and immune response was examined when the antigen (A/Indonesia/5/2005 (H5N1)) supplemented with ampligen at 20-fold ratio of HA antigen, and CVP was transnasally administered 1 - 4 times, using, as a comparison control, an antigen+Ampligen+CVP transnasal administration group for the first half of the test group and transnasal administration of antigen alone/intramuscular administration of antigen alone/saline transnasal administration for the latter half. The results are shown in Tables 12-19 and Fig. 3.
  • In the antigen+Ampligen+CVP group, several to dozen-fold mucous membrane IgA/serum IgG and NT antibody titer were observed as compared to the antigen alone transnasal administration group, and the difference was remarkable in the groups with lower dose and less administration frequency.
  • Conventional common knowledge holds that, as compared to vaccines for subcutaneous or intramuscular administration, transnasal administration-type vaccines show lower specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer. However, the vaccine evaluated at this time was found to acquire specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer equivalent to or above those of the vaccines with conventional composition and administration method, by the addition of AmpligenĀ® and the like.
  • Furthermore, the immune response of the samples obtained by this test with the same strain as the administered antigen and a strain with different antigenicity was examined to confirm cross-reactivity.
    Figure imgb0001
     [Table 11]
    Vaccine composition
    dose administration group vaccine stock solution(ĀµL) Ampligen (ĀµL) C\/P (ĀµL) M/75PBS(pH72) (ĀµL) total (mL) note
    high dose (0.3Āµg HA/dose) 1,4,7,10 302.42 2400.0 3000 297.58 6 for 1000 times (transnasal)
    moderate dose (0.1Āµg HA/dose) 2,5,8,11 100.81 800.0 3000 2099.19 6 for 1000 times (transnasal)
    low dose (0.033Āµg HA/dose) 3,6,9,12 33.27 264.0 3000 2702.73 6 for 1000 times (transnasal)
    high dose (0.3Āµg HA/dose) 13,16,19,22 302.42 5697.58 6 for 1000 times (transnasal)
    moderate dose (0.1Āµg HA/dose) 14,17,20,23 100.81 5899.19 6 for 1000 times (transnasal)
    low dose (0.033Āµg HA/dose) 15,18,21,24 3327 5966.73 6 for 1000 times (transnasal)
    high dose (0.3Āµg HA/dose) 25,26,27,28 3024 4969.76 5 for 100 times (muscular injection)
    saline 29 600 (saline) for 100 times (transnasal)
    Whole particles influenza vaccine: A/Indo/5/2005(H5N1) lot No.; FPBMW0612-c (protein concentration: 3110 Āµg/mL, HA content 992 Āµg HA/mL)
    Ampligen (manufactured by Hemispherx): lot; 0701HE (containing 2.5 mg/mL Ampligen)
    CVP base (manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya): lot INF-N28; carboxyvinyl polymer 1.1%, L-arginine 2.4%, glycerol 2%
    Figure imgb0002
    Figure imgb0003
    Figure imgb0004
     [Table 15]
    Serum HI test (2009.7.23) cross-reaction (A/VN)
    group 4 5 7 8 26 27
    antigen amount of administration (Āµg HA/dose) 0.3 0.1 0.3 0.1 0.3 0.3
    administration frequency 3 2 3 2
    administration route transnasal transnasal transnasal transnasal intramuscular intramuscular
    adjuvant (Ampligen), CVP addition yes yes yes yes none none
    animal No. 1 5 20 5 5 5 5
    2 5 5 5 5 5 5
    3 5 5 5 5 20 5
    4 5 5 5 5 10 5
    5 5 10 5 5 5 5
    6 5 5 5 5 5 5
    7 5 20 5 5 5 5
    8 5 20 5 5 5 5
    9 5 5 5 10 5 5
    10 5 5 5
    average antibody titer (GMT) 5.0 8.6 5.0 5.4 6.2 5.0
    variance 0.0 7.3 0.0 1.7 4.8 0.0
    seroconversion rate (antibody titer not less than 40,%) 0Ā·0 0.0 0.0 0.0 0.0 0.0
    Figure imgb0005
    Figure imgb0006
    Figure imgb0007
    Figure imgb0008
    • (Example 3) Administration of transnasal administration-type influenza vaccine (H5N1 type) and immune response evaluation test using Cynomolgus monkeys (Macaca fascicularis)
  • Based on the relationship between the administration dose, use and immune response obtained by the experiments using mice in Example 2, administration tests were performed using Cynomolgus monkeys (Macaca fascicularis) as an animal close to human, and the dosage form and composition optimal as a transnasal administration-type vaccine were searched and the usefulness from the aspect of infection defense was investigated.
  • To be precise, (1) search of optimal dosage form/composition by comparison of immune responses after administration of transnasal administration-type vaccine candidates, and (2) evaluation of practicality from the ability to defend against infection by measurement of serum and mucous membrane antibody titers after vaccine inoculation were performed.
  • The search was performed by immuno-chemical test of serum (influenza HI antibody titer, IgG-ELISA antibody titer and neutralizing antibody titer) and immuno-chemical test of nasal swab (influenza specific IgA-ELISA antibody titer).
    • (Test method) 1. Influenza vaccine stock solution:
  • Influenza vaccine (H5N1 strain) stock solution (lot No., FPBMQI0813, protein concentration: 816 Āµg/mL, HA content 324 Āµg HA/mL, origin of virus strain: A/Bar-headed
    Goose/Qinghai/1A/2005 (H5N1))
    • 2. Origin of virus strain and inactivated vaccine antigen to be used for test (for specific antibody titer measurement)
  • A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) strain: attenuated strain (clade 2.2) of A/Qinghai/1A/2005 (H5N1)
    • (for cross-reactive antibody titer measurement)
    • A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2 strain: attenuated strain (clade 2.1) of A/Indonesia/5/2005 (H5N1)
    • A/Anhui/01/2005 (H5N1)/PR8-IBCDC-RG5 strain: attenuated strain (clade 2.3) of A/Anhui/01/2005 (H5N1)
    • NIBRG-14 strain: attenuated strain (clade 1) of A/Vietnam/1194/2004 (H5N1)
    3. Adjuvant and additive to be used for test
    • Ampligen: manufactured by Hemispherx, (containing 10.61 mg/mL of Ampligen)
    • carboxyvinyl polymer (CVP): manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya, lot; INF-619 (containing 1.1% CVP, 2.4% L-arginine and 2% glycerol)
    • poly L-arginine: manufactured by SIGMA, catalog No. P-3892, lot; 107K5103 (MW>70,000)
    4. Test method
  • Fig. 4 shows outline of test flows. [Table 20]
    Test group constitution:
    group vaccine HA antigen (Āµg/dose) Ampligen (Āµg/ dose) Poly L-Arg (Āµg/ dose) CVP addition head note
    1 30 - 4 vaccine alone
    2 30 600 O 4 trial vaccine
    3 30 1500 O 4 Poly L-Arg
    4 - - 1 control group
  • Anesthesized Cynomolgus monkeys (Macaca fascicularis) was transnasally administered with a vaccine containing A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) strain virus antigen or saline at 3 weeks intervals, using a transnasal administration device with trial actuator (test actuator : manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya, Spray Pump; Apta Pharma, VP-7 type, spraying use) six times by 150 Āµl (50 ĀµlƗ3) to each nasal cavity (total 300 Āµl), and samples (nasal swab and serum) were taken at the time of each administration, and every 2 weeks from the booster up to 12 weeks from the booster. Furthermore, where necessary, blood samplesā€¢nasal swab were taken every 2 weeks thereafter.
    • Animal and vaccine:
    • animal: Cynomolgus monkeys (Macaca fascicularis) (male 17 cynomolgus monkeys, 46 - 58 months of age when test was started, body weight 2.8 - 3.5 kg in November 2009), group 1 - group 4: 4 cynomolgus monkeys/group, group 5 (negative control): cynomolgus monkey/group
    • trial vaccine for test and adjuvant dose:
      1. (1) non-addition vaccine group: [30 Āµg HA]/dose
      2. (2) Ampligen + CVP addition vaccine group: [30 Āµg HA + 600 Āµg Ampligen + 0.55% CVP]/dose
      3. (3) Poly L-arginine + CVP addition vaccine group: [30 Āµg HA + 0.5% Poly L-arginine + 0.55% CVP]/dose
      4. (4) negative control group: saline
    vaccine administration volume: 150 Āµl for each nasal cavity, total 300 Āµl 5. Measurement items of each sample
  • nasal swab: specific IgA-ELISA antibody titer and IgA concentration
  • The nasal swab was measured for the total IgA concentration and the specific IgA-ELISA antibody titer calculated in the same manner as in Example 2 was amended to be the numerical value per total IgA concentration of 1 Āµg/mL. serum: specific IgG-ELISA antibody titer, specific, cross-reactive HI (hemagglutination inhibition) antibody titer, specific/cross-reactive neutralizing antibody titer
  • Among the above-mentioned measurement items, the outline of the measurement method of the ELISA antibody titer was as described below.
  • Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to protein concentration of 1 Āµg/mL) was solid phased (4Ā°C, overnight) on a 96-well ELISA plate at 100 ĀµL/well, and washed 3 times with PBS containing 0.1% Tween 20. A sample diluted 2-fold series or negative control was added at 100 ĀµL/well. Then, the plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. An antibody for detection (alkaliphosphatase-labeled anti-monkey IgG or biotin-labeled anti-monkey IgA) was added at 100 ĀµL/well. The plate was incubated at 37Ā°C for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. The substrate solution (4-NPP or TMB) was added at 100 ĀµL/well and the mixture was shaded and incubated at room temperature for 30 min.
  • A stop solution (2M H2SO4 solution for 4-NPP or 650 nm stop solution for TMB) was added, and the absorbance (405 nm or 655 nm) was measured.
  • The maximum dilution rate that affords an absorbance of sample exceeding the average absorbance of negative control+2SD was taken as the antibody titer of the sample. For specific antibody titer, the same strain as the administration vaccine antigen was used and, for cross-reactive antibody titer, vaccine antigen of a different strain was used. In the following, HI antibody titer and neutralizing antibody titer were measured in the same manner.
    specific HI (hemagglutination inhibition) antibody titer and cross-reactive HI antibody titer: They were measured in the same manner as in Example 2.
    specific neutralizing antibody titer and cross-reactive neutralizing antibody titer: They were measured in the same manner as in Example 2.
    • (Results)
  • The results are shown in Tables 21-30 and Figs. 5-7.
  • It was confirmed that transnasal spray administration of a vaccine containing Ampligen and CVP to monkey induces specific IgA production in the mucous membrane, and derivation of specific neutralizing antibody in the serum. Since these effects were insufficient with an additive-free vaccine, the usefulness of addition of Ampligen and CVP was confirmed. [Table 21]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test using Cynomolgus monkeys (Macaca fascicularis);
    Vaccine composition
    group vaccine HA antigen (mL) [324 Āµg HA/mL] Ampligen (mL) [10.61 mg/mL] Poly L-Arg (mL) [100 mg/mL] CVP addition (mL) M/75 PBS (pH 7.2) (mL) Note dose (ĀµL)
    1 3.087 (30 Āµg/dose) - - - 6.913 10 mL min 300
    2 3.087 30 Āµg/dose) 1.885 (600 Āµg/dose) - 5.0 (yes) 0.028 10 mL min 300
    3 - - 0.750 (1.5 mg/dose) - 4.250 5 mL min 100
    3.704 (30 Āµg/dose) - - 4 0.296 8 mL min 200
    4 - - - 10.000 10 mL min 300
    Influenza vaccine stock solution to be used for test
    influenza vaccine (H5N1 strain) stock solution: lot No.; FPBMQI0813
    (protein concentration: 816 Āµg/mL, HA content 324 Āµg HA/mL)
    origin virus strain: A/Bar-headed Goose/Qinghai/1A/2005 (H5N1)
    adjuvant and additive to be used for test
    Ampligen (manufactured by Hemispherx): containing 10.61 mg/mL of Ampligen
    carboxyvinyl polymer (manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya): lot; INF-619 (containing 1.1% CVP (carboxyvinyl polymer), 2.4% L-arginine and 2% glycerol)
    poly L-arginine (manufactured by SIGMA, catalog No. P-3892): lot; used after dissolving 107K5103 (MW>70,000) in 100 mg/mL
    [Table 22]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); before administration (blood samples were collected on 2010.1.28/measured on 2010.2.2)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 5 5 5 5
    2 5 5 5
    3 5 5 5
    4 5 5 5
    average antibody titer (GMT) 5.0 5.0 5.0 5.0
    variance 0.0 0.0 0.0
    seroconversion rate (antibody titer not less than 40, %) 0.0 0.0 0.0 0.0
    [Table 23]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); after once administration (blood samples were collected on 2010.2.18/measured on 2010.2.23)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 20 5 5 5
    2 10 5 5
    3 5 5 5
    4 5 10 5 5
    average antibody titer (GMT) 8.4 5.9 5.0 5.0
    variance 7.1 2.5 0.0
    seroconversion rate (antibody titer not less than 40, %) 0.0 0.0 0.0 0.0
    [Table 24]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); after twice administration (blood samples were collected on 2010.3.4/measured on 2010.3.9)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 5 5 5 5
    2 5 5 5
    3 5 5 5
    4 5 5 5
    average antibody titer (GMT) 5.0 5.0 5.0 5.0
    variance 0.0 0.0 0.0
    seroconversion rate (antibody titer not less than 40, %) 0.0 0.0 0.0 0.0
    [Table 25]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); 2 weeks after twice administration (blood samples were collected on 2010.3.18/measured on 2010.3.24)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 5 5 5 5
    2 5 5 5
    3 5 5 5
    4 5 5 5
    average antibody titer (GMT) 5.0 7.1 5.9 5.0
    variance 0.0 2.9 2.5
    seroconversion rate (antibody titer not less than 40, %) 0.0 0.0 0.0 0.0
    [Table 26]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (NT) using Cynomolgus monkeys (Macaca fascicularis); before administration (blood samples were collected on 2010.1.28/measured on 2010.2.2)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 5 10 5 5
    2 5 10 20
    3 5 5 10
    4 5 5 20
    average antibody titer (GMT) 5.0 7.1 11.9 5.0
    variance 0.0 2.9 7.5
    [Table 27]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (NT) using Cynomolgus monkeys (Macaca fascicularis) after NT administration once (blood samples were collected 2010.2.18/measured on 2010.2.23)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 5 10 20 5
    2 10 20 5
    3 20 20 10
    4 5 160 140
    average antibody titer (GMT) 8.4 28.3 19.3 5.0
    variance 7.1 71.8 64.5
    [Table 28]
    Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (NT) using Cynomolgus monkeys (Macaca fascicularis) after NT administration twice (blood samples were collected on 2010.3.4/measured on 2010.3.9)
    group 1 2 3 4
    vaccine HA antigen (Āµg/dose) 30 30 30 -
    Ampligen (Āµg/dose) 600
    Poly L-Arg (Āµg/dose) 1500
    CVP addition - + + -
    animal No. 1 20 20 80 5
    2 40 160 5
    3 40 160 80
    4 10 1280 40
    average antibody titer (GMT) 23.8 160.0 33.6 5.0
    variance 15.0 587.1 36.1
    [Table 29]
    Serum IgG ELISA titer after administration of transnasal administration-type influenza vaccine (H5N1-type) using Macaca fascicularis
    group 1 2 3 4
    antibody titer Pre 141.4 168.2 200.0 100.0
    Post-3w 336.4 1131.4 237.8 50.0
    Post-5w 951.4 4525.5 1600.0 200.0
    Post-7w 400.0 5381.7 800.0 100.0
    [Table 30]
    Nasal swab IgA ELISA titer after administration of transnasal administration-type influenza vaccine (H5N1-type) using Cynomolgus monkeys (Macaca fascicularis)
    group 1 2 3 4
    antibody titer Pre 0.7 1.5 1.4 1.1
    Post-3w 0.9 1.6 1.3 0.5
    Post-5w 0.7 4.4 2.9 0.6
    Post-7w 2.9 4.3 6.5 0.6

Claims (12)

  1. A vaccine composition for nasal mucosal administration, comprising (i) an influenza virus antigen, (ii) polyriboinosinic polyribocytidylic acid (poly (I:C)), poly (I:CxU) or poly (IxU:C), and (iii) a carboxyvinyl polymer, wherein x is on average a number from 3 to 40.
  2. The composition of claim 1, wherein the antigen is a subunit antigen or an inactivated antigen.
  3. The composition of claim 2, wherein the subunit antigen comprises at least one subunit antigen selected from the group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza virus.
  4. The composition of claim 2, wherein the subunit antigen comprises at least one subunit antigen selected from the group consisting of HA and NA.
  5. The composition of claim 1, wherein the poly (I:CxU) is poly (I:C12U).
  6. A vaccine composition comprising an effective amount of (i) an influenza virus antigen, (ii) poly (I:C), poly (I:CxU) or poly (IxU:C), and (iii) a carboxyvinyl polymer, wherein x is on average a number from 3 to 40, said composition being for use in a method of preventing influenza, comprising a step of administering said composition at least once to the nasal mucosa of a subject in need thereof.
  7. The composition for use according to claim 6, wherein the antigen is a subunit antigen or an inactivated antigen.
  8. The composition for use according to claim 7, wherein the subunit antigen comprises at least one subunit antigen selected from the group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza virus.
  9. The composition for use according to claim 7, wherein the subunit antigen comprises at least one subunit antigen selected from the group consisting of HA and NA.
  10. The composition for use according to claim 6, wherein the poly (I:CxU) is poly (I:C12U).
  11. The composition for use according to claim 6, wherein the method comprises administering the composition at least twice.
  12. The composition for use according to claim 11, wherein the method comprises administering the composition at an interval of at least 1 week.
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